The present invention relates to a novel hybridoma cell line, in particular it relates to a novel hybridoma cell line which secretes monoclonal antibodies capable of binding to the AT1 subtype of the Angiotensin II receptor (AT1 receptor). The invention also relates to monoclonal antibodies secreted by the hybridoma, which antibodies may be used in a test kit having several diagnostic and monitoring applications. It also relates to the use of the monoclonal antibodies in therapeutic applications such as the control of smooth muscle stimulation.
The hormone angiotensin II (Ang II) forms part of the reninxe2x80x94angiotensin system which helps to control electrolyte balance and blood pressure within the body. There are several tissues within the body upon which Ang II acts, they include the adrenal gland, uterus, liver, brain and kidney.
Amongst the several established functions of angiotensin II, it is known to stimulate smooth (unstriated) muscle cell contraction. It stimulates the contraction of smooth muscle cells in the blood vessel wall thus causing vaso-constriction, which leads to hypertension. Most treatments for high blood pressure will include blockage of angiotensin function in one way or another. Smooth muscle also occurs in other locations, for example in the uterus and in the gastrointestinal tract, and elsewhere.
Ang II also stimulates the secretion of aldosterone by the adrenal cortex. Aldosterone is a potent hormone which acts primarily on the kidney to promote sodium retention and thus inter alia, heightens the hypertensive effects of angiotensin acting directly on the vasculature.
Ang II is known to act on various sites in the brain, and one of its actions in animals is the regulation of thirst and drinking.
Angiotensin also has trophic effects on the vasculature, promoting growth of the muscles in the arterial wall. It is also thought to be angiogenic, i.e. it causes vascularisation of newly developing tissue.
The actions of angiotensin II in cells are mediated through two important intracellular signalling mechanisms. When the hormone binds to its receptor, it activates a specific enzyme, phospholipase C, which acts upon a constituent of the cell membrane called phosphatidyl inositol. This is split by the enzyme into two moieties, called inositol trisphosphate (IP3), and diacylglycerol (DAG). Both of these are involved in eliciting further effects within the target cell. IP3 stimulates increased cellular cytosolic calcium concentrations, which in turn evokes other cellular responses, whereas DAG stimulates another specific enzyme called protein kinase C (PKC).
Most of the established effects of Ang II have been found to occur via the AT1 subtype of the Ang II receptor, which is a seven transmembrane domain receptor. This receptor has been cloned and sequenced from a variety of tissues, and has been found to be a 359 amino acid polypeptide with a predicted molecular weight of around 40 kD (Bernstein and Alexander, (1992), Endocr. Rev., 13, 381-386). Studies using photo-affinity labelling and crosslinking agents have suggested molecular weights for mature receptor of approximately 65 kD and 116 kD, respectively, which may reflect glycosylation of asparagine residues within the extra-cellular domain.
Whilst polyclonal antibodies and anti-idiotypic antibodies have been prepared to the Ang II receptor, from which it has been postulated that the receptor has a molecular weight of from 60 to 95 kD, and 63 kD respectively, to date no-one has succeeded in preparing a monoclonal antibody to this receptor.
It has now been found that by immunising mice with a synthetic peptide corresponding to amino acid residues 8-17 of the rat vascular smooth muscle AT1 receptor (Murphy, T. J. et al, (1992), Nature, 351, 233-236), and thereafter fusing spleen cells from the immunised mice with mouse myeloma cells, a novel hybridoma cell line is produced, which secretes monoclonal antibodies to the AT1 subtype of the Ang II receptor.
According to one aspect of the invention there is provided a hybridoma cell line which produces monoclonal antibodies capable of binding to the AT1 subtype of the angiotensin II receptor. A hybridoma cell line that secretes such monoclonal antibodies was deposited on 22 Jul. 1993 with the European Collection of Animal Cell Cultures, Porton Down, United Kingdom, under the Budapest Treaty, and designated accession No. 93072117.
Such a hybridoma cell line produces antibodies which bind specifically to amino acid residues 8 to 17 of the rat vascular smooth muscle AT1 receptor. It was found, however, that the monoclonal antibodies would bind to the AT1 receptor in bovine and human tissue, as well as in rat tissue. To date this sequence of amino acid residues has been found, and is therefore conserved, in all mammalian AT1 receptors so far cloned. Thus the hybridoma cell line produces antibodies that bind specifically to a peptide having the following amino acid sequence: (SEQ. ID NO. 1).
xe2x80x94H2N-Glu-Asp-Gly-lle-Lys-Arg-Asp-COOHxe2x80x94
According to a second aspect of the invention there is provided a monoclonal antibody that binds to the AT1 subtype of the angiotensin II receptor. Such monoclonal antibodies bind specifically to amino acid residues 8-17 of the mammalian AT1 receptor, that is they bind specifically to a peptide having the amino acid sequence given above.
A hybridoma cell line, according to the invention, can be prepared by immunising inbred mice by techniques well known in the art (Kohler and Milstein, (1975), Nature, 256, 495-497). A peptide was synthesised which corresponds to amino acid residues 8-17 (extracellular) of the published rat vascular smooth muscle AT1 receptor. The peptide was then conjugated to bovine serum albumin (BSA) and used to immunise mice.
Following a booster injection of the peptide-BSA conjugate the mouse spleens were removed, and the spleenocytes were combined with mouse myeloma cells. Mixed myelomaxe2x80x94lymphocyte hybrids were selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium.
The presence of hybridoma cell lines producing monoclonal antibodies to the AT1 subtype of the angiotensin II receptor was first detected by screening the hybridoma conditioned medium for binding to a rat liver cell suspension. Such positive binding was detected using peroxidasexe2x80x94conjugated rabbit anti-mouse immunoglobulin (IgG) antibody. Following the initial screening, cell cultures showing positive results were expanded and tested for specific binding to rat adrenal glomerulosa on both frozen sections and dispersed cell smears, using a fluorescein-conjugated rabbit anti-mouse (IgG) antibody. The integrity of the anti-AT1 receptor monoclonal antibodies produced by the hybridoma was confirmed by binding to, rat AT1A receptor transiently expressed by transfected Cos-7 cells.
The monoclonal antibodies of the present invention may be tagged with compounds that fluoresce at various wavelengths, so that the location and distribution of AT1 receptors in body tissues can be determined by immunohistological techniques. For example, using this monoclonal antibody AT1 receptors have been found in breast tumours, thus the antibody may be useful in the cancer diagnostic field.
In addition, using the monoclonal antibody of the present invention a hitherto unknown site of action for Ang II has been discovered. It has been found that both rat and human sperm tails express the angiotensin II receptor, and the physiology of the regulation of its expression leads to the possibility that the hormone may be profoundly important in the control of sperm motility, with a potential effect on male fertility. The monoclonal antibodies of the present invention may thus be used in a standard radioimmuno-assay or enzyme-linked immunosorbant assay to study, and possibly measure, sperm motility, as well having a use in contraception.
According to a further aspect of the invention there is provided a diagnostic test kit comprising the monoclonal antibodies of the present invention attached to a detectable label. Such detectable labels include radioisotopes, enzymes and fluorescent compounds.
It has been found that addition of the monoclonal antibodies according to the present invention to living cells inhibits the angiotensin II-generated IP3 response, but it has no action on PKC activation. Thus the antibody interacts highly specifically with just one of the two major signalling pathways stimulated by angiotensin II. This property can be used experimentally to discriminate between the effects of these two pathways, as well as in therapeutic applications. For example, the monoclonal antibodies may be used in controlling vaso-constriction and therefore used to treat hypertension, as well as for the regulation of menstruation and the control of uterine contractions to prevent miscarriage.
Furthermore these monoclonal antibodies can be used in both immunohistochemistry and immunoelectron-miscroscopy, and may have applications in immunoblotting and immunocytological staining.
The invention is illustrated by the following example.